Effective Preparation of Plasmodium vivax Field Isolates for High-Throughput Whole Genome Sequencing

Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss a methodology to significantly improve the success of P. vivax WGS on natural (non-adapted) patient isolates. Using 37 patient isolates from Indonesia, Thailand, and travellers, we assessed the application of CF11-based white blood cell filtration alone and in combination with short term ex vivo schizont maturation. Although CF11 filtration reduced human DNA contamination in 8 Indonesian isolates tested, additional short-term culture increased the P. vivax DNA yield from a median of 0.15 to 6.2 ng µl⁻¹ packed red blood cells (pRBCs) (p = 0.001) and reduced the human DNA percentage from a median of 33.9% to 6.22% (p = 0.008). Furthermore, post-CF11 and culture samples from Thailand gave a median P. vivax DNA yield of 2.34 ng µl⁻¹ pRBCs, and 2.65% human DNA. In 22 P. vivax patient isolates prepared with the 2-step method, we demonstrate high depth (median 654X coverage) and breadth (≥89%) of coverage on the Illumina GAII and HiSeq platforms. In contrast to the A+T-rich P. falciparum genome, negligible bias was observed in coverage depth between coding and non-coding regions of the P. vivax genome. This uniform coverage will greatly facilitate the detection of SNPs and copy number variants across the genome, enabling unbiased exploration of the natural diversity in P. vivax populations.     

Target-Dependent Enrichment of Virions Determines the Reduction of High-Throughput Sequencing in Virus Discovery

Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.     

Universal Count Correction for High-Throughput Sequencing

We show that existing RNA-seq, DNase-seq, and ChIP-seq data exhibit overdispersed per-base read count distributions that are not matched to existing computational method assumptions. To compensate for this overdispersion we introduce a nonparametric and universal method for processing per-base sequencing read count data called Fixseq. We demonstrate that Fixseq substantially improves the performance of existing RNA-seq, DNase-seq, and ChIP-seq analysis tools when compared with existing alternatives.     

一株辛德毕斯病毒的基因组序列测定及分析

目的：对引进的一株辛德毕斯病毒的基因组序列进行测定,阐明其与已报道毒株序列的关系。方法：对辛德毕斯病毒基因组编码区进行分段RT-PCR扩增,对非编码区采用RACE法进行扩增,将扩增产物直接进行测序,应用DNAStar软件将测序结果拼接得到基因组序列,采用MEGA3.1软件对9株辛德毕斯病毒基因组序列进行系统进化发生树的构建。结果与结论：此株辛德毕斯病毒基因组共11663nt,编码3745个氨基酸残基,其中5＇端的2/3基因组编码4种非结构蛋白NSp1、NSp2、NSp3和NSp4,3＇端的1/3基因组编码5种结构蛋白E1、E2、E3、6K和C;结构基因和非结构基因之间有48nt的连接区为非翻译区;病毒基因组5＇末端和3＇末端分别有59、318nt的非编码区;序列同源性分析结果表明,此株病毒与S.A.AR86株的同源性最高,两者核苷酸序列的同源性为99.7%,氨基酸序列的同源性为99.6%,而与本室保存的另一辛德毕斯病毒MEI株的遗传进化关系稍远,系统进化发生树处于不同分支上。     

Evolution of the Influenza A Virus Genome during Development of Oseltamivir Resistance In Vitro

Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.     

The Genome-Packaging Signal of the Influenza A Virus Genome Comprises a Genome Incorporation Signal and a Genome-Bundling Signal

The influenza A virus genome comprises eight single-stranded negative-sense RNA segments (vRNAs). All eight vRNAs are selectively packaged into each progeny virion via so-called segment-specific genome-packaging signal sequences that are located in the noncoding and terminal coding regions of both the 3′ and the 5′ ends of the vRNAs. However, it remains unclear how these signals ensure that eight different vRNAs are packaged. Here, by using a reverse genetics system, we demonstrated that, in the absence of the other seven vRNAs, a recombinant NP vRNA bearing only a reporter gene flanked by the noncoding NP regions was incorporated into virus-like particles (VLPs) as efficiently as a recombinant NP vRNA bearing the reporter gene flanked by the complete NP packaging signals (i.e., the noncoding sequences and the terminal coding regions). Viruses that comprised a recombinant NP vRNA whose packaging signal was disrupted, and the remaining seven authentic vRNAs, did not undergo multiple cycles of replication; however, a recombinant NP vRNA with only the noncoding regions was readily incorporated into VLPs, suggesting that the packaging signal as currently defined is not necessarily essential for the packaging of the vRNA in which it resides; rather, it is required for the packaging of the full set of vRNAs. We propose that the 3′ and 5′ noncoding regions of each vRNA bear a virion incorporation signal for that vRNA and that the terminal coding regions serve as a bundling signal that ensures the incorporation of the complete set of eight vRNAs into the virion.     

一株丙型肝炎病毒缺陷株NS5A基因片段的序列分析

Ｑ丙型肝炎是由丙型肝炎病毒（ＨＣＶ）引起的一种严重的传染病。丙型肝炎病毒主要通过输血和应用不洁的血液制品传播,所以利用敏感、特异的检测方法筛选献血员对丙型肝炎的预防尤为重要。现在第二代ＨＣＶ检测试剂已大批应用,加入ＮＳ５Ａ抗原的第三代试剂也正在研制中...     

一株保存的波瓦生病毒全基因组序列测定及分析

目的：对保存的WJBC株波瓦生病毒进行全基因组序列测定和分析,阐明其与已报道毒株之间的关系。方法：将波瓦生病毒基因组编码区分11段进行RT－PCR扩增,扩增产物直接进行测序,非编码区采用RACE法进行扩增,扩增产物纯化并连接pGEM－Teasy载体后转化大肠杆菌DH5ct感受态细胞,挑取阳性克隆鉴定后进行测序,用DNAstar软件将测序结果拼接得到全基因组序列。下载波瓦生病毒全基因组核苷酸序列,利用MEGA5．0软件构建系统进化发生树。结果与结论：WJBC株波瓦生病毒全基因组共11839nt,编码3415个氨基酸残基,病毒基因组5’端和3’端分别有111、483nt的非编码区;进化树结果显示,WJBC株波瓦生病毒与LB株波瓦生病毒的亲缘性最高,可能为同一病毒株．．     

Strategy for Modular Tagged High-Throughput Amplicon Sequencing

The use and validation of a strategy that allows a universal set of bar-coded sequencing primers to be appended to an amplified PCR product is described. The strategy allows a modular approach, in that the same bar code can be used with two or more target-specific primer sets, even simultaneously.     

香蕉束顶病毒基因Ⅰ的克隆及序列分析

以中国漳州地区感染ＢＢＴＶ的香蕉组织总ＤＮＡ为模板,根据我国台湾地区ＢＢＴＶ分离物基因组Ⅰ序列,设计并合成了一对引物,通过ＰＣＲ扩增出约５００ｂｐ的片段。利用ｐＢｌｕｅｓｃｒｉｐｔⅡＳＫ Ｔ－载体获得此片段的克隆,经测序表明为ＢＢＴＶ组分Ⅰ的部分序列。由已测知的ＢＢＴＶ基因组Ⅰ序列设计一对相邻引物,以我国漳州的感染ＢＢＴＶ香蕉组织总ＤＮＡ为模板,通过ＰＣＲ坟增出约１．１ｋｂ的片段。利用ｐＢｌｕｅｓ     

Systematic Identification of H274Y Compensatory Mutations in Influenza A Virus Neuraminidase by High-Throughput Screening

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.     

Complete Genome Sequencing of Four Geographically Diverse Strains of Batai Virus

Batai virus (BATV) is a widely distributed but poorly studied member of the Orthobunyavirus genus in the family Bunyaviridae and is of particular interest as a known participant in natural reassortment events. Both research and surveillance efforts on this and other related viruses have been hampered by the lack of available full-length sequence data covering all three genomic segments. Here, we report the complete genome sequence of four BATV strains (MM2222, Chittoor/IG-20217, UgMP-6830, and MS50) isolated from various geographical locations. Based on these data, we have determined that strain MS50 is in fact unrelated to BATV and likely represents as a novel genotype in the genus Orthobunyavirus.     

Method for Increasing the Regularity of Isolation of Infectious Ribonucleic Acid from Influenza Virus

A more effective method of isolating infectious ribonucleic acid (RNA) from influenza virus was evaluated based on the enzymatic disintegration of the viral coat by Pronase, followed by phenol-detergent extraction of the RNA from susceptible and from resistant cells.     

Inhibitors of the Influenza A Virus M2 Proton Channel Discovered Using a High-Throughput Yeast Growth Restoration Assay

The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A and L26F in the pore of the channel. Despite progress in designing inhibitors of V27A and L26F M2, there are currently no drugs targeting these mutated channels in clinical trials. Progress in developing new drugs has been hampered by the lack of a robust assay with sufficient throughput for discovery of new active chemotypes among chemical libraries and sufficient sensitivity to provide the SAR data essential for their improvement and development as drugs. In this study we adapted a yeast growth restoration assay, in which expression of the M2 channel inhibits yeast growth and exposure to an M2 channel inhibitor restores growth, into a robust and sensitive high-throughput screen for M2 channel inhibitors. A screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified 21 active compounds comprising amantadine, rimantadine, 13 related adamantanes and 6 non-adamantanes. Of the non-adamantanes, hexamethylene amiloride and a triazine derivative represented new M2 inhibitory chemotypes that also showed antiviral activity in a plaque reduction assay. Of particular interest is the fact that the triazine derivative was not sufficiently potent for detection as an inhibitor in the traditional two electrode voltage clamp assay for M2 channel activity, but its discovery in the yeast assay led to testing of analogues of which one was as potent as amantadine.